In-vitro Antitumor Activity and Antifungal Activity of Pennogenin Steroidal Saponins from paris Polyphylla var. yunnanensis.

Paris polyphylla Smith var. yunnanensis, has been used in traditional Chinese medicine for its antibiotic and anti-inflammatory properties; in addition it has been used to cure liver cancer in particular. In this current study, β-ecdysterone (1) and three pennogenin steroidal saponins (2-4) were isolated from the EtOH extract of Paris polyphylla var. yunnanensis, and then tested for their antitumor and antifungal activities. Spectroscopic data was used to confirm their structures. Their antitumor properties were determined by using an MTT assay in addition to ethidium bromide and acridine orange staining techniques. Compounds 2, 3 and 4 exhibited significant anti-proliferation activities against HepG2 cells, with IC50 values of 13.5 μM, 9.7 μM and 11.6 μM respectively, obtained following 48 h treatment. Furthermore, we found these pennogenin steroidal saponins could induce HepG2 cells apoptosis at a concentration of 20 μM after 48 h treatment. Compounds 2, 3 and 4 were confirmed to exhibit moderate antifungal activity. The minimum inhibitory concentration (MIC) of compounds 2, 3 and 4 against saccharomyces cerevisiae hansen were 2.5 mg.mL(-1), 0.6 mg.mL(-1) and 0.6 mg.mL(-1), respectively. The MIC of compounds 2, 3 and 4 against Candida albicans were 1.2 mgmL(-1), 0.6 mg.mL(-1) and 1.2 mg.mL(-1), respectively. The analysis of the bioactivity-structure relationship shows that the sugar moiety plays a critical role in the activity of steroid moiety. Our results suggest that these three pennogenin steroidal saponins could be utilized to develop anticancer medicines.


Introduction
Medical herbs have been used for a few thousands years and are considered to be one of the most promising sources of new medicines and leading compounds due to their therapeutic effects demonstrated in clinical use. Paris polyphylla Smith var. yunnanensis, used as a Chinese traditional medicine, is widely distributed in China. It has been widely used due In addition this herbal medicine has been used to treat liver cancer in particular (1). Certain steroidal saponins isolated from paris polyphylla have been reported to convey antitumor and immuno-stimulating properties (2-4). This paper reports the isolation, structure determination and biological activities of -ecdysterone and three pennogenin steroidal saponins (2-4). We found proliferation activities against HepG2 cells and induced apoptosis of HepG2 cells.

Isolation
The dried aerial part of Paris polyphylla var. yunnanensis, purchased from the Western Medicine City of Chongqing, China, were twice evaporated to achieve the ethanol extract. The ethanol extract was partitioned successively with chloroform, ethyl acetate and n-butanol.

Data of isolated compounds
The spectra data of compounds 1-4 were measured: NMR on a 500 MHz Bruker DRX-500 instrument (Bruker, German) and MS on a VG Auto-spec3000 mass spectrometer (VG, England).

MTT assay
The MTT assay was performed according to the method set out by Mosmann(5). The HepG2 cells were plated into 96-well microtiter plates at a density of 1×10 4 cells/well. After 24 h, the 1640 medium supplemented with 10% fetal bovine serum containing varying concentrations 4. The cells were then subjected to incubation for was less than 0.1% in the cell culture medium. The culture solutions were removed and replaced to each well. The absorbance at 490 nm of the dissolved solution was measured using a Bio-Rad 680 microplate reader (BIORAD, USA). The relative cell viability (%) of the control wells containing cell culture medium without the tested compound was calculated by dividing the absorbance of treated cells by that of the controls in each experiment. The IC 50 was calculated as the tested compound concentration by means of SPSS statistical software, which inhibits the growth of 50% of cells in relation to non-treated control cells.

AO/EB staining assay
The cells were cultured on coverslips and kept in a 60 Petri dish for 24 h before treatment. Following treatment for 48 h with compounds then immediately (less than 20 mins) observed JAPAN).

Assay for antifungal activity
The antifungal activity against saccharomyces cerevisiae hansen and candida albicans of compounds 1-4 was evaluated by determining their minimum inhibitory concentrations (MIC), using broth microdilution techniques.
The MIC values were determined in RPMI-1640 (Hyclone, USA). Stock solutions of pure compounds were twofold diluted with RPMItest tubes. Each tube was then inoculated with 6 determined by using the optical density of the solutions. The lowest concentration of drug that inhibited all fungal growth was determined as being the MIC.

Results and Discussion
The EtOH extract of paris polyphylla var. yunnanensis was suspended in H 2 O and fractionated using chloroform, ethyl acetate and n-butanol, successively. The n-butanol soluble fraction, which showed major inhibitory activity on HepG2 cells, was repeatedly subjected to silica gel column chromatography and Source 15 RPC column chromatography to afford compounds 1-4. The structures of compounds 1-4 were analyzed using 1 H, 13 C NMR and MS.

Antitumor activity
Two experiments were carried out to compounds.
The potential anti-proliferative effect of these cells was evaluated by means of the MTT assay.
inhibiting the growth and proliferation of HepG2 cells (data not shown). Cell viability is expressed as the mean percentage (±SD) of viable cells in comparison with untreated cells (taken as 100% viable) for different concentrations of compounds inhibited the growth and proliferation of HepG2 cells in a dose-and time-dependent manner ( Figure 2). The cell viability was shown to be 12.32%, 6.48% and 9.6% following treatment respectively. The estimated IC 50 (50% of growth is inhibited) value of compounds 2, 3 and 4 software.
To further address the cell death pattern, HepG2 cells after treatment for 48 h with compounds 2, 3 and 4 at a concentration of 20 ethidium bromide (AO/EB), and immediately EB staining combines the differential uptake EB and the morphologic aspect of chromatin condensation within the stained nucleus. This allows viable, apoptotic and necrotic cells to be distinguished from each another. Viable cells possess a uniformly bright green nucleus. Early apoptotic cells show bright green areas of condensed or fragmented chromatin within the nucleus. Necrotic cells exhibit a uniformly bright orange nucleus. After staining with AO/ EB, the HepG2 cells showed a slight change in cell morphology with a very rough periphery. Effervescence, crumb-like structures, and nuclear fragmentation occurred, apoptotic bodies appeared, the permeability of cell membrane increased, and a window for both stained AO and EB was observed, which is a typical apoptosis characteristic (Figure 3). We believe that the apoptotic bodies visualized in our results are a cell apoptosis process within cancerous cells by treatment with either of the compounds and not just a mere overall toxic effect of the chemicals.

Antifungal activity
The antifungal test of the compounds from paris polyphylla var. yunnanensis gave the results listed in Table 2. The results presented as minimal inhibitory concentration (MIC, ) show that compounds 2, 3 and 4 convey relatively higher antifungal activities against saccharomyces cerevisiae hansen and candida albicans than compound 1. The MIC of compounds 2, 3 and 4 against saccharomyces cerevisiae hansen -1 -1 -1 , respectively. The MIC of compounds 2, 3 and 4 against Candida albicans -1 -1 -1 , respectively.

Structure-activity relationships
Compounds 2, 3 and 4, with the same steroidal saponin moiety, have different anticancer and antifungal activities. Compound 3 exhibits the strongest activity and also has the most sugar moieties among the three. This experimental and control value.

Concenttration (μM)
Cell Viability (%) A B C may suggest that there is a relation between the anticancer and antifungal activities of these compounds and the number of sugar moieties they contain. The water-solubility of these compounds increases in correlation with an increase in their sugar moiety number. Their anticancer and antifungal activity decreases in the order: compound 3 (4 sugar moieties) > compound 4 (3 sugar moieties) > compound 2 (2 sugar moieties). The hetero-sugar moiety causes the hetero-polarity of these compounds leading to different membrane permeability and selectivity, involved in the bioactivity of the compounds. The bioactivity-structure relationship of the compounds containing more sugar moieties than four remains unclear. Too many sugar moieties may lead to an excessively large polarity, which would then impair the solubility of the active ingredient within the lipophillic medium of the biomembrane and thus block its transmembrane transfer. Sugar moiety has been reported to play a critical role in the activity of steroid moiety. Diosgenin, stigmasterol and solanidine, which have the same skeleton and different sugar moieties, show hetero-activity in the cell cycle arrest (10). OSW-1, characterized as a cholestane disaccharide, is an extraordinarily potent antitumor saponin. Whereas, the glycon OSW-1 and the disaccharide OSW-1 show proliferation of tumor cells (11). These facts all allude to the importance of a precise number of sugar moieties in the bioactivity of saponins within living cells.

Conclusion
In this work, -ecdysterone (compound 1) and three other compounds with the same steroidal saponin moiety were isolated from Paris polyphylla var. yunnanensis and their compounds 2, 3 and 4 not only inhibited cancer cell proliferation but in addition induced cancer cell apoptosis. These three pennogenin steroidal saponins may provide clues for designing a range of novel semi-synthetic and synthetic compounds as medicinal anti-cancer agents in the near future.